This invention provides methods for the identification of novel biological targets and/or inhibitors. Such methods provide useful and convenient tools for high throughput screening. In particular, such methods may be used to identify novel antimicrobial agents.
The emergence of resistance to current therapeutic agents, such as antibacterial, antifungal, and antimalarial agents, necessitates the development of novel agents. Whilst target-based biochemical screens can be successful, classic anti-microbial screening has clearly outperformed rational target-selecting procedures in terms of discovered novel antimicrobials. Target-based biochemical screens are frequently seen to invoke a lack of cell activity, despite a good inhibition of the target enzyme, perhaps due to low permeation into or export out of the cell. Also the presence of inhibiting structures for a hand-picked target within a limited set of available molecules is by no way guaranteed.
In contrast, cell-based antimicrobial screening frequently identifies antimicrobial action but then either reveals this inhibition to be caused by general toxicity, for example membrane perturbation or DNA-intercalation, or fails to identify any specific interaction of the drug with the cellular machinery. This complicates chemical optimisation of initial leads, providing no guide for a structure-activity relationship.
A particular approach to identify target-based mode of action has been that of hypersensitivity.
Historically, the way to generate hypersensitive versions of a protein of interest has been the generation of temperature-sensitive (ts) mutations in its encoding gene (Schmid et al. Genetics 123, pp625-633, (1989)). Temperature sensitive (Ts) or other conditional phenotypes can be generated by either targeted (for example using PCR) or random (for example using chemicals or radiation) mutagenesis. However, such conditional mutants are frequently hypersensitive to inhibitors even under permissive conditions.
We have now found that hypersensitivity to inhibitors can be readily achieved by shutting down a particular essential function of viable cells followed by analysis of the effects in the presence/absence of a potential inhibitor. By xe2x80x9cshutting downxe2x80x9d we mean that the particular essential function is not available to the cells at any expression level.
Therefore in a first aspect of the present invention we provide a method for identifying a compound which modulates the function of the gene product of an essential gene, which method comprises providing viable cells wherein the gene is expressed under the control of a heterogeneous, regulatable promoter, switching off gene expression via the promoter, contacting the cells with a test compound and determining any modulatory effect on the function of the gene product.
The method provides a step jump in the feasibility of a complete genome analysis for compound hypersensitivity. It provides a number of significant advantages not least that the unaltered xe2x80x9cnormalxe2x80x9d protein is used in contrast to for example extrapolation from the interaction of an altered, mutagenized protein with an inhibitor onto the interaction of the same chemical. Also no effort is needed to fine tune promoter activity to a higher or lower level. Inhibition of one specific biochemical entity, for example a protein required for cellular growth or survival is conveniently indicated by a hypersensitive response of the strain with the regulatable gene compared to its unaltered parent.
By xe2x80x9cheterogeneous, regulatable promoterxe2x80x9d we mean a promoter other than the native promoter for the essential gene and which can be regulated by the addition and/or removal of specific materials or for example by other environmental changes.
By xe2x80x9chypersensitivityxe2x80x9d we mean a larger reduction in cell growth in the presence of an identical concentration of drug, compared to a wild-type cell.
Furthermore, hypersensitivity caused by underexpression of one specific enzyme can extend to a whole biochemical pathway, such as for example sterol biosynthesis. This is exemplified by the results shown in FIG. 5 of this invention, where the sensitivity to terbinafine is altered both by shutdown of the target enzyme (encoded by ERG1) as well as shutdown of another enzyme (encoded by ERG11) in the same pathway.
By xe2x80x9cviablexe2x80x9d we mean cells that have grown sufficiently so that when gene expression of an essential gene is turned off, meaningful measurements and kinetic analysis may be made. The cells are preferably allowed to grow into early stationary phase by which time a final optical density reading is taken. A reduced optical density compared to no drug or no switchoff controls is interpreted as growth inhibition.
By xe2x80x9cessential genexe2x80x9d we mean a gene required by a cell for example for cell growth and/or cell viability. This does include genes required only under certain assayable conditions (ie conditional essential). In its simplest manifestation essentiality is defined as necessary for growth of the organism on rich media (For a comprehensive summary of such media as well as general yeast methodology see Sherman et al in Methods in Enzymology, Vol 194, Guthrie and Fink eds, Academic Press (1991). Convenient genes include fungal and bacterial genes.
By xe2x80x9cswitching off gene expressionxe2x80x9d we mean that all gene expression is turned from ON to OFF. There is no low level gene expression in the OFF position.
By xe2x80x9ccellsxe2x80x9d we mean cells from any convenient source, these include human, animal or microbial cells. New genome-based techniques are developing which combine screening of compounds with simultaneous target identification. Whilst this invention is of particular use with genetically tractable model organisms such as Schizosaccharomyces pombe and Saccharomyces cerevisiae, it is universally applicable and is limited only by practical considerations. Using model organisms such as S. cerevisiae, S. pombe, C. elegans, or D. melanogaster it is possible to study conserved functions of eukaryotes. The application of genetics using human or animal cell lines directly, then extends possibilities further.
Any convenient test compound such as a peptide, nucleic acid and low molecular weight compound, may be used in the methods of the invention. Preferred test compounds are potential therapeutic agents or may be used in further studies to identify therapeutic agents. Particular test compounds are low molecular weight compounds of, for example, molecular weight of less than 1000, such as less than molecular weight 600.
The modulatory effect of a compound on the gene product of an essential gene is conveniently investigated taking endpoint optical density readings (the higher the absolute optical density of the culture, the less inhibition is thought to have occured. Such analysis is conveniently effected over period of typically 24 hours for yeast cells. Bacteria may take significantly shorter (eg 12 hours) whereas higher eukaryotic cells will require several days to reach early stationary growth phase. The analysis is conveniently photometric (optical density of the culture at 600 nm wavelength).
Importantly, the combination of very recent techniques for site specific integration of DNA (for example with a switchable promoter directly replacing the native one) with the observed fixed timepoint optical density analysis of the hypersensitive response (as further elaborated below) is novel and allows exploitation for large-scale drug screening, compound and target profiling. Moreover, such strains are also very easily obtainable in very high quantity (ie. thousands of genes in S. cerevisiae).
Some examples of switchable promoters for use in S. cerevisiae include MET3 (repressible by added methionine) and GAL1 (repressed by glucose induced by galactose); for use in S. pombe: NMT1 (repressed by thiamine); for use in C. albicans: MAL1 (repressed by glucose, induced by maltose, sucrose); for use in E. coli: araB (repressed by glucose, induced by arabinose); for use in Gram-positive bacteria such as Staphylococci, Enterococci, Streptococci and Bacilli: xylA/xylR (from S. xylosus) (repressed by glucose, induced by xylose); for use in E.coli and B.subtilis pSPAC (an artificial promoter derived from E.coli lac, regulated by IPTG, see Vagner et al. Microbiology (1998), 144, 3097-3104); and for all of the above organisms plus further unspecified fungi, bacteria and mammalian cell lines: tetA/tetR (from various bacterial tetracycline resistance cassettes) this system exists in various versions, see Gossen et al. Current Opin. Biotechnol. 5, pp516-520 (1994), that are repressible or inducible by various tetracycline analogues.
We illustrate in the Example and Figures below that it is possible to generate hypersensitivity towards any agent that inhibits a biochemical function by placing the gene encoding that function (protein or RNA) under control of a tightly regulatable promoter and switching it from the ON to the OFF state whilst simultaneously adding the interacting substance (for illustration of such a switch see FIG. 1). Whilst it is known to place external promoters in front of genes of interest to achieve over- or under-expression of the encoded protein, this process requires labour-intensive fine-tuning and setting up. Due to strongly varying expression levels, every gene of interest would require a different tuning of its promoter strength to be at a just growth-rate-limiting level. Therefore such a protocol relying on fine-tuning is unlikely to allow implementation at the high throughput scale necessary for genomic analysis.
Our novel findings, as detailed in the experiments shown below, demonstrate that a simple and standard protocol using just one promoter at clearly defined ON and OFF states (ie. explicitly without any finer modulation of the expression level) is capable to generate accurate measurements of specific hypersensitivity due to target underexpression. The underexpression is caused by i) decay of the RNA, ii) decay of the target protein, iii) dilution of the target molecule into several daughter cells during cell growth in the absence of any resynthesis.
The examples below demonstrate, for example, that unknown fungal target proteins that can be inhibited by compounds can be identified by placing their essential genes under control of a regulatable promoter, switching it off and exposing it to compound levels that do not inhibit the corresponding wild type cell. Currently 814 S. cerevisiae genes have been shown essential for growth even on rich media, (source YPD(trademark).) An observed hypersensitive response (eg in a fungus such as S. cerevisiae) shows: i) the target is essential ii) the compound is an antifungal agent or a starting point for evolution of a more potent analogue iii) the matching target-compound pair (or pairs) establishes mode of action for the antifungal compound. For more than one compound detected against one target, valuable structure-activity relationship (SAR) information can be obtained. Comparison and pattern recognition analysis of many compound effects on many targets will establish a valuable database useful for grouping novel targets into pathways immediately. Hypersensitivity also allows the target-specific detection of low potency compounds not identifiable using the parental strain.
A further advantage of the invention is that characterised biochemical activity of the target molecule is not a pre-requisite for the methods of the invention. Hypersensitivity as such can provide an assayable feature intrinsically linked with a specific target. Genomic switchoff constructs can be generated and integrated into the organisms genome in very high throughput using published PCR-based methodology without the involvement of any cloning step (Longtine et al. Yeast, 14, pp 953-961 (1998); Zhang et al. Nature Genetics, 20, pp 123-128 (1998). Therefore the methods of the invention may be used to test as many compounds as possible against every potential antifungal target, for example up to 10, up to 20, up to 50, up to 100, up to 500, or up to 1000 targets using modern high throughput screening technology.
Therefore in a further method of the invention we provide a method for identifying metabolic pathway drug hypersensitivity which method comprises providing viable cells wherein a gene in the metabolic pathway is expressed under the control of a heterogenous, regulatable promoter, switching off expression of the gene via the promoter, contacting different groups of the cells with different test compounds, determining and comparing any modulatory effect on the growth of the viable cells. Underexpression of one element of a biochemical pathway and chemical inhibition of another part has been shown to be synergistic (see FIG. 5) thus generating pathway specific information by underexpressing just one element of that pathway.
The invention will now be illustrated but not limited by reference to the following Example and Figures wherein:
FIG. 1 shows the generation of a controllable allele of gene, Gen1, by replacing its native promoter with a well defined and tight ON/OFF switch; here GAL1
FIG. 2 shows Erg11p activity in wild type (wt) and GAL1-ERG11 strains and illustrates how catalytic activity declines both with time and increased concentration of an inhibitor. The culture is shifted from galactose to glucose at time 0, whereby new synthesis of Erg11p (lanosterol C-14-demethylase) is stopped. The fluconazole concentration is sub-inhibitory (for example 10 mg/l) for a wild type (wt) strain.;xe2x80x94xe2x80x94:no fluconazole, . . . : fluconazole; the threshold is where Erg11p activity is rate-limiting for growth.
FIG. 3 shows the growth of wild type (JK9-3da, Kunz et al. Cell, 73, pp585-596(1993)), GAL1-ERG11 and GAL1-AUR1 (generated as described in Longtine et al. Yeast, 14, pp 953-961 (1998))strains in the presence of fluconazole;
FIG. 4 shows the growth of wt, GAL1-ERG11, and GAL1-AUR1 strains in the presence of aureobasidinA;
FIG. 5 shows the growth of wt, GAL1-ERG11, and GAL1-AUR1 strains in the presence of terbinafine;
FIG. 6 shows a protocol for promoter replacement in S. cerevisiae (substantially as described in Longtine et al. Yeast, 14, pp 953-961 (1998)).